Quantification of bone marrow interstitial pH and calcium concentration by intravital ratiometric imaging

Abstract The fate of hematopoietic stem cells (HSCs) in the bone marrow can be directed by microenvironmental factors including extracellular calcium ion concentration (T h can vary significantly with bone remodeling, but the local e around individual HSCs in vivo remains unknown. Here we developed an intravital ratiometric imaging approach to quantify the absolute pH and in the mouse calvarial bone marrow, taking into account the pH sensitivity of the calcium probe and the wavelength-dependent optical loss through bone to ensure unbiased ratiometric analyses. We uncovered substantial heterogeneity in f (1.0 - 3.6 mM) surrounding steady-state HSCs. While the lowest a (0.3 mM) was found in regions dominated by bone formation, HSCs were not found in those locations. This work thus established a tool to further investigate t and pH in the HSC niche under malignant or stressed conditions and can be broadly applied to other tissue types.